developing the logistics for producing human pathogen-free organic strawberries in the state of...
DESCRIPTION
2014 National Sustainable Strawberry Initiative Project Leader MeetingTRANSCRIPT
![Page 1: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/1.jpg)
Developing the
Logistics for
Producing Human
Pathogen-Free
Organic Strawberries
in the State of
Tennessee
A project funded by a grant from the Walmart Foundation and administered by the University of
Arkansas System Division of Agriculture Center for Agricultural and Rural Sustainability
![Page 2: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/2.jpg)
Participants
PIs: Drs: Suping Zhou,
Fur-chi Chen,
Roy-Bullock,
Theodore W Thannhauser.
Research associates and assistants:
Long Zhang,
Christine Moore,
Kathyleen Yan,
Himabindu Gazula (graduate student).
Eight local farms in Middle Tennessee.
![Page 3: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/3.jpg)
0
2000
4000
6000
8000
10000
12000
14000
16000
1950'S 1970'S 2010'S
Acreage
Acreage in Strawberries over the years
• 1950’s: Tennessee was the #1 state in the United States for growing strawberries
using a matted row system with about 15,000 acres
• 1970’s: Acreage dropped to about 200 acres due to labor, disease (anthracnose), and
other factors
• 2010’s: Current growing practices utilize around 575 acres
• Plasticulture with approximately 500 acres
• Matted Row approximately 50-75 acres
• Organic production approximately 10 acres
*Reference: Bob Ary, County Extension Agent for Sumner County, TN
Background information of the project*
![Page 4: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/4.jpg)
Outbreaks of human pathogen on
fresh and frozen strawberries have
been reported on
E. coli O157:H7, Salmonella,
hepatitis A,
Listeria monocytogenes
Incidences of human pathogen contamination of fresh strawberries
![Page 5: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/5.jpg)
Project Goal and Priority
•Goal is to promote sustainable fresh strawberry production in Middle Tennessee
•Two priority areas in this project:1) Reduce the risk of human pathogens on fresh berries;2) Implement meaningful and constructive metrics for strawberry production sustainability.
![Page 6: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/6.jpg)
Objective 1. Developing detection method for human pathogens on fresh strawberries
•Identification of microbial populations on organic and inorganic fresh strawberries.•Survival analysis of the human pathogens in fresh strawberry.•Developing dipstick assay method to detect the human pathogens on fresh strawberries
![Page 7: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/7.jpg)
• In July, 2013, strawberry samples collected from local farms,and immediately inoculated with bacterial strains of
•Escherichia coli 0157:H7
•Solmonella typhimurium
•Listeria monocytogenes.
•Strawberries were stored at 4 and 25 oC for two months and thenthese human pathogens were analyzed
Detection of human pathogens on
strawberries (juice) after long-term storage
![Page 8: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/8.jpg)
1.Direct spreading and
2.Enrichment method
Identification of pathogenic bacteria
Fig 3:Direct spreading
Fig 4:Enrichment method
Results: Non-bacterial
colony was recovered,
indicating all bacteria
were died.
![Page 9: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/9.jpg)
1 2
75Kda
10 Kda
Lane 1: Strawberry sample with E. coli/Listeria
Lane 2: Bacterial Control of E. Coli/Listeria
66.2Kda66 Kda
1 2
Western blot analysis
E.coli Listeria sppFig 5: Fig 6:
![Page 10: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/10.jpg)
Western Blot Analysis of Salmonella Typhimurium
Lanes 1 and 3: Strawberry extract with S.typhimuriumLanes 2 and 4: Bacterial Control of S.typhimurium
1 2 34
Monoclonal studyPolyclonal study
Results Cont…
Fig 7:Fig 8:
Results:
Bacteria were
died, but their
proteins were
still intact in
strawberry
juice.
![Page 11: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/11.jpg)
Procedure of detection
Growing of isolated cultures
of bacteria in tryptic soy broth
Spot inoculation of strawberry with
10µl of suspended bacteria
Storage of strawberries at
4˚C and 25˚C
Homogenization in 50 mL butterfield
buffer by stomacher
Serial dilution of homogenate in 9
mL butterfield buffer
Spread plating on selective media
plate
Analysis of bacterial colonies
Fig 9: Spot inoculation of strawberry
with bacteria
![Page 12: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/12.jpg)
Growth of bacteria on fresh strawberries
0
2
4
6
8
10
Day One Day Three Day Five
E.coli
X-axis: Day of culture
Y-axis: Log CFU/berry
4°C
25°C
0
1
2
3
4
5
6
Day One Day Three Day Five
Listeria
0
1
2
3
4
5
Day One Day Three Day Five
Salmonella
![Page 13: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/13.jpg)
Objective 2 Developing Dip-Stick Detection of Single Bacterial Colony on Fresh strawberries
Detection methods:
E. coli 0157:H7 Reveal 2.0 medium,
Salmonella: Rappaport-vassiliadis (RV) medium,
Listeria: Listeria Enrichment Single Step (LESS) medium
Fig.20 Dip Sticks
“Dip stick analysis” method has been followed
to detect human pathogens viz. E. coli
(0157:H7), Salmonella typhimurium and
Listeria monocytogenes in fresh strawberry.
![Page 14: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/14.jpg)
Dip stick analysis procedure
Serial dilutions of bacterial culture
Inoculation of strawberry with 100µl diluent from each dilution.
Incubation at selective temperature for 24-48 hrs.
Observation of (single) colonies in the selective media plates
Dip stick analysis
![Page 15: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/15.jpg)
Results
Dip stick analysis of Listeria
Single bacterial colony of Listeria Dip stick analysis of Listeria
![Page 16: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/16.jpg)
Results
Dip stick analysis of Salmonella
Single bacterial colony of Salmonella Dip stick analysis of Salmonella
![Page 17: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/17.jpg)
Results
Single bacterial
colony of E. coli
Agglutination test showing
the presence of E. coli
Dip stick analysis of E. coli 0157:H7
![Page 18: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/18.jpg)
Conclusion
The Dipstick assay can produce reliabledetection result for single colony inoculationof Salmonella and Listeria on freshstrawberries;
Detection of E. coli 0157:H7 through thecommercially available dip sticks isn’tsuccessful. So, there is a need to develop animprovised tool to detect the presence ofE.coli on contaminated food products.
![Page 19: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/19.jpg)
Objectives: Three and Fourth
Develop science based modules for
sustainable organic, human-pathogen- free
strawberry production through outreach
activities
![Page 20: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/20.jpg)
1. McCraw’s Farms
2. Head Farms
3. Madison Creek Farms
4. Wade Farmz
5. Southland Farms
6. Mountain View Farms
7. Ferrell Farms
8. Pickett Farms
8 farms located in the Middle Tennessee:
![Page 21: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/21.jpg)
Ground Breaking
Soil Sampling
Planted Strawberries
Soil analysis:
•Five farms were very high in phosphorus
and potassium
•One farm was low in phosphorus, but a
different farm was low in potassium
•Other farms were high in phosphorus and
contained a medium amount of potassium.
• Calcium and magnesium were sufficient
for strawberry growth
*Analysis completed by the UT Extension Soil, Plant, and Pest
Center in Nashville, TN
![Page 22: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/22.jpg)
Farm Management
• Fungal and Bacterial Controls*• Oxidate
• Hydrogen Dioxide – 27.1%
• Peroxyacetic Acid – 2.0%
• Acitovate AG• Streptomyces lydicus WYEC 108
• Pest Control*• Trilogy
• Clarified Hydrophobic Extract of Neem Oil – 70%
• Weed Control*• Hand Weed
• AXXE• Broad Spectrum Herbicide
• Ammonium Nananoate – 40%
• Soil Analysis from each plot/field
• Soil Amendments*• BioVam – T&J Enterprises
• Endomycorrhizae, Ectomycorrhizae, and additional Biological community
• SeaFish Liquid Fertilizer• 5-1-1
• whole Menhaden (ocean) fish
• Green Potash• Soluble Potash (K2O) 0-0-15 (without
sulfur)
• Derived from Greensand and Kelp
* All amendments and control additives OMRI approved
* Amendment selection through consultation with extension agents and purchasing entities
![Page 23: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/23.jpg)
A booming production 3 of the 5 farms with 2 other farms in question
McCraw’s Farm
Head’s Farm
2 gallons picked from
1 demonstration plot
inoculated with
BioVam
Wade Farmz
2 ½ gallons picked from 1
demonstration plot on the
most eastern edge of the
fieldAlmost 3 gallons with
largest amount
harvested from the
BioVam plot (Back
Plot)
![Page 24: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/24.jpg)
BioVam’s Advantage
Without BioVam With BioVam
![Page 25: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/25.jpg)
Identification of human pathogen contamination points
• Irrigation water;
• Domestic animals;
• Humans on U-pick
farms.
![Page 26: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/26.jpg)
Outreach activities
1. Workshop: One at TSU;
2. Blind taste survey, >200;
3. Portland (Tennessee) Strawberry Festival, >500 visitors;
4. Demonstrations in public events, four times;
5. Social media and websites, >2000 visitors.
![Page 27: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/27.jpg)
![Page 28: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/28.jpg)
![Page 29: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/29.jpg)
Portland Strawberry FestivalsMay 10, 2014
![Page 30: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/30.jpg)
![Page 32: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/32.jpg)
![Page 33: Developing the Logistics for Producing Human Pathogen-Free Organic Strawberries in the State of Tennessee](https://reader034.vdocument.in/reader034/viewer/2022042715/5584253ed8b42ac0658b4782/html5/thumbnails/33.jpg)
Next activity
Strawberry Variety comparison test Six strawberry varieties harvested from the project demonstration plots will be compared in a blind taste test